PROJECT SUMMARY Alcohol use disorder (AUD) is a major health concern affecting millions of Americans with an estimated annual cost of well over $200 Billion. A critical component in limiting the impact of AUD is preventing relapse, a common tribulation for alcoholics. Stress dramatically increases the likelihood of reinstatement during protracted abstinence, and patients with AUD often have comorbid anxiety disorders. Modulation of the endocannabinoid (eCB) system can decrease the response to stressors. The insula and BNST have emerged as regions implicated in the withdrawal phase of AUD. Evidence suggests that the insula plays a vital role in the negative interoceptive stimuli experienced during abstinence. The BNST, part of the extended amygdala, integrates reward and stress in addiction. Our lab has found functional monosynaptic connections between the insula and anxiety-responsive corticotrophin-releasing factor (CRF) cells in the BNST (BNSTCRF cells). The inputs that control the BNST projecting insular (insulaBNST) neurons and the modulation of those inputs remain essential unknowns. The proposed research aims to understand the regulation of the insulaBNST circuit and how it is changed in ethanol withdrawal. To do so, we will further investigate the distribution of afferents to insulaBNST cells (subaim 1.1), establish monosynaptic connectivity between afferent regions (subaim 1.2), and interrogate ethanol and eCB functionality at these synapses (subaim 1.3). Subaim 2.1 will ascertain the effects of alcohol withdrawal on ex vivo slice physiology in this circuit while subaim 2.2 will establish the implications of alcohol withdrawal in vivo using calcium imaging via fiber photometry. We hope the findings from these experiments will provide a better understanding of the interplay between these brain regions and how this interaction is regulated. In so doing, we will be able to establish the normal physiology and impact that alcohol withdrawal has on this novel circuit.